Fig. 2: CBP β-hydroxybutyrylates p53 inside cells and in vitro.

a p53 kbhb in the presence or absence of BHB in cells. Western blot analysis of p53. IP was performed using BHB-K antibodies in cells transfected with p53 or both p53 and CBP and then treated with 10 mM BHB. b CBP mutants lacked the ability to exert kbhb activity on p53. Western blot analysis of p53 performed using antibodies against BHB-K, Ac-K and specific acetylated sites on p53 in IP obtained from cells transfected with p53, p53 plus wild-type CBP, or p53 plus mutant CBP and then treated with 10 mM BHB, 1 μM TSA and 5 mM nicotinamide for 24 h. c CBP β-hydroxybutyrylates p53 in vitro. Purified p53 was incubated with CBP or CBP mutants and β-hydroxybutyrate-CoA (BHB-CoA). Western blot analysis of the reaction products performed with BHB-K antibodies (top two panels), with anti-p53 (p53 labeled panel) or with anti-HA antibody (bottom panel to detect CBP). d Silencing CBP reduced the β-hydroxybutyration level of p53 in U2OS. Cells was transfected twice with 120 pmol of siRNA oligos specially targeting CBP and p300 or negative control, followed by treating with 10 mM BHB after 48 h. e p300 β-hydroxybutyrylates p53 in vitro. Purified p53 was incubated with p300 and BHB-CoA. Western blot analysis of the reaction products performed with BHB-K antibodies (top two panels), with anti-p53 (p53 labeled panel) or with anti-Flag antibody (bottom panel to detect p300)